ascorbic-acid and sodium-thiocyanate

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu2+ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm.Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-AsA system, although hydrogen peroxide (H2O2) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H2O2 serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu2+ or Cu+) at the specific site of inactivation.

Topics: Ascorbic Acid; Benzoylarginine Nitroanilide; Catalase; Free Radical Scavengers; Histidine; Hydrogen Peroxide; Organometallic Compounds; Oxidation-Reduction; Papain; Potassium Iodide; Reactive Oxygen Species; Spectrophotometry, Ultraviolet; Substrate Specificity; Thiocyanates; Thiourea

We determined the effects of phenol and 2,6-dimethoxyphenol (syringol) on N-nitrosomorpholine (NMOR) formation in rats given morpholine and nitrite by gavage. At 30 min post-gavage the recovery (from the stomach, duodenum and blood) of 564 micrograms NMOR was six times higher when administered to rats by gavage with 2 g of semipurified diet (SPD) than when given without food. Rats were gavaged with 12 mg each of morpholine, one of the modifiers and nitrite and examined 30 min later. Syringol decreased the amount of NMOR in both the stomach and blood by 89%, while phenol had no effect. We compared these results with those obtained with ascorbic acid and thiocyanate. The effect of ascorbic acid was similar to that of syringol. However, thiocyanate increased the amount of NMOR in the stomach and blood 2.7- and 4-fold, respectively. When 2 g of SPD was administered to rats by gavage, together with the precursors, syringol and ascorbic acid blocked NMOR formation in the stomach by 58 and 45%, respectively, and thiocyanate enhanced the yield 1.5-fold. The effect of phenol was not significant for the stomach and blood and that of the other modifiers was not significant for blood. Administration of the reactants together with food decreased the NMOR level in blood 155-fold relative to controls (no food), suggesting that food decreased the absorption rate over a 30-min period. These results demonstrate the modifying effect of phenol and syringol on NMOR formation in vivo to be similar to that observed in a previous in vitro study, and show that the effect of food on NMOR levels in blood was more important than that of the modifiers.

Topics: Animals; Ascorbic Acid; Food; Food, Formulated; Gastric Mucosa; Gastrointestinal Contents; Intestinal Absorption; Male; Morpholines; Nitrites; Nitrosamines; Phenol; Phenols; Pyrogallol; Rats; Rats, Inbred Strains; Thiocyanates